Nitrogen is one of the pillars of worldwide agricultural production. Many have argued that improving the efficiency with which fertilizer nitrogen is used in world agriculture is essential to the long-term sustainability of the planet.
The amino acid glutamine (Gln) is a critical intermediate in nitrogen metabolism across all life. In plants, nitrogen fertilizer is assimilated into Gln (12, 39) and subsequently glutamate; together, these amino acids serve as nitrogen donors for various reactions, including the biosynthesis of other amino acids (34, 39). These amino acids further regulate uptake and assimilation of soil nitrate and ammonium (13, 25, 29, 40, 61). Gln, as well as asparagine, are important forms of nitrogen used for long-distance transport in the plant, including from root to shoot (49). In fact, the application of nitrogen fertilizer to the roots of maize (Zea mays L.) plants can generate a large increase in the levels of Gln and other amino acids within 30-120 min in the root and shoot, respectively (34). Gln is thus a key indicator of nitrogen status in plants. Unfortunately, only indirect or delayed assays currently exist to quantify plant nitrogen status, in spite of the importance of nitrogen as the most limiting soil nutrient in global agriculture (55).
Free Gln is primarily quantified using high performance liquid chromatography (HPLC)-based analysis of tissue extracts (1, 38, 43, 47). There has been limited use of biosensors for Gln quantification. The Gln-binding protein (QBP) (32), originating from the periplasmic space of bacteria, has been used to construct reagentless sensors for Gln for use with aqueous extracts (14, 15). For cell biology applications, a Förster resonance energy transfer (FRET) QBP-based biosensor has been engineered in transgenic Arabidopsis plants to monitor Gln uptake by root tips under a fluorescent microscope (63). This biosensor achieves millimolar sensitivity with superb sub-cellular resolution and good specificity for Gln, but requires the creation of transgenic plants which may be less suited for high throughput applications, including determination of plant nitrogen status.
Microbial whole cell biosensors have been used as inexpensive tools to quantify analytes from biological extracts into which they are co-inoculated (18, 50). They have also been used to visualize analytes in intact tissues of non-transgenic organisms. For example, the leakage of sucrose, tryptophan or iron can be sensed in plants using nearby microbial biosensor cells and then imaged (27, 33).
A subset of microbial biosensors have been based on auxotrophs expressing a constitutive reporter (e.g. green fluorescent protein, GFP, lux). An auxotroph is an organism that cannot grow in the absence of a particular metabolite. The use of amino acid auxotrophs for the quantification of bioavailable essential amino acids such as lysine in human and animal foodstuffs originated decades ago (6, 7, 18, 26, 48, 60). A Gln auxotroph of Escherichia coli has been generated by down-regulation of the gene encoding glutamine synthetase (GlnA) (36).
Most of the currently available methods for glutamine measurement have the disadvantages of being expensive, time consuming and requiring some level of technical expertise. Furthermore, conventional methods are indirect or delayed. It is therefore desirable to provide compositions, media, and methods for detecting glutamine that mitigate at least one of the problems or disadvantages of the prior art.